Enhanced Heterologous Spinosad Production from a 79-kb\nSynthetic Multioperon Assembly

Refactoring\nbiosynthetic pathways for enhanced secondary metabolite\nproduction is a central challenge for synthetic biology. Here we applied\nadvanced DNA assembly methods and a uniform overexpression logic using\nconstitutive promoters to achieve efficient heterologous production\nof the complex insecticidal macrolide spinosad. We constructed a 79-kb\nartificial gene cluster in which 23 biosynthetic genes were grouped\ninto 7 operons, each with a strong constitutive promoter. Compared\nwith the original gene cluster, the artificial gene cluster resulted\nin a 328-fold enhanced spinosad production in <i>Streptomyces\nalbus</i> J1074. To achieve this goal, we applied the ExoCET\nDNA assembly method to build a plasmid from 13 GC-rich fragments with\nhigh efficiency in one step. Together with our previous direct cloning\nand recombineering tools, we present new synthetic biology options\nfor refactoring large gene clusters for diverse applications.